Use of a neutral or cationic polymer to prevent to activation of blood or plasma in contact with a semi-permeable membrane

ABSTRACT

The invention relates to the use of a neutral or cationic polymer for preventing activation of the contact phase of blood or plasma which comes into contact with a semi-permeable membrane based on polyacrylonitrile bearing fixed negative charges, which is fitted in apparatus for the treatment of blood or plasma by extra-corporal circulation, by combination of the neutral or cationic polymer, before or after formation of the membrane and before its sterilization, so as to modify the electrical properties of the membrane, the Zeta potential “Z” and the electrical resistivity “R” in order to obtain: 
     either an electrokinetic index “I” of less than or equal to 0.8, “I” corresponding to Log 10  (|Z|/R), or 
     a positive Zeta potential “Z” of between 0 and 15 mV (limits not included).

The present invention relates to the use of a neutral or cationicpolymer, combined, before sterilization, with a semi-permeable membranebased on polyacrylonitrile bearing fixed negative charges, which isfitted in apparatus for the treatment of blood or plasma byextra-corporal circulation, to prevent activation of the contact phaseof blood or plasma.

The subject of the present invention is also apparatus for the treatmentof blood or plasma by extra-corporal circulation, which preventsactivation of the contact phase of blood or plasma, as well as processesfor manufacturing this apparatus.

Apparatus for the treatment of blood or plasma by extra-corporalcirculation is used in various medical or paramedical applications, suchas: treatment of renal insufficiency by dialysis or haemofiltration,plasmapheresis and apheresis for therapeutic and non-therapeuticpurposes, oxygenation of the blood, immunopurification, etc.

Activation of the contact phase of blood or plasma appears to take placein particular when apparatus for the treatment of blood or plasma byextra-corporal circulation is used, which includes a negatively-chargedsemi-permeable membrane, without, in the absence of disruptive factors,the patients experiencing the slightest discomfort. Activation of thecontact phase is described as a biological phenomenon which takes placein the case of patients undergoing blood (or plasma) treatment byextra-corporal circulation, when the blood comes into contact with thenegatively charged surface of the membrane of certain apparatus forblood and plasma treatment. This biological phenomenon results in thegeneration of active substances, kallicrein and factor XIIa frominactive substances, prekallicrein and factor XII, kallicrein having acatalytic effect on the production of factor XIIa, and vice versa. Inaddition, kallicrein is the cause of the transformation of a plasmaprotein, molecular weight kininogen, into a peptide substance,bradykinin.

When activation of the contact phase takes place simultaneously withcertain disruptive factors such as, for example:

the presence, in the blood to be treated, of medicinal products forcombating arterial hypertension by inhibition of the natural mechanismof vasoconstriction, these products being referred to generically asconversion enzyme inhibitors, or CEIs. These CEIs are also used forother therapeutic applications, in particular for treating certain formsof cardiac insufficiency,

dilution of the blood entering the apparatus filled with saline solutionand concomitant lowering of the blood pH,

activation of the contact phase appears to be the cause of adversereactions called anaphylactoid.

These anaphylactoid reactions develop, a few minutes after the start ofthe treatment, by various symptoms, including a generalized sensation ofhotness, swelling of the fingers, the lips or the tongue, gasping forbreath, nausea and laryngeal oedema.

Anaphylactoid reactions have been observed in particular in the case ofrenal insufficiency patients treated by haemodialysis, haemofiltrationor haemodiafiltration with the aid of apparatus for the treatment of theblood in the form of a dialyser or exchanger containing a membrane.

Anaphylactoid reactions have been observed with exchangers containingmembranes of various chemical compositions, sometimes during a firstuse, sometimes after several uses when the exchangers, rather than beingdiscarded after a single use, are reused several times and recycledafter each use. As examples of exchangers for which a first use has beenaccompanied by an adverse reaction, mention may be made of dialyserscontaining a membrane based on polymethyl methacrylate and onpolyacrylonitrile. Reactions associated with the reuse of dialyserscontaining a membrane based on cellulose acetate and on polysulphonehave also been well documented (see the article entitled “Anaphylactoidreactions associated with reuse of hollow-fiber haemodialysers and ACEinhibitors” in “Kidney International”, vol. 42 (1992), pp. 1232-1237).

Anaphylactoid reactions are ascribed to an excessive concentration ofbradykinin in the blood or in the plasma.

In order to avoid the generation of bradykinin at a concentration above4000 pg/ml, European patent No. 0,561,379 recommends placing the bloodor the plasma in contact only with semi-permeable membranes which have alimited surface charge density, i.e. an overall surface electricalcharge of greater than or equal to −30 μeg/g of membrane, thiselectrical charge being measured by a method chosen from the groupconsisting of the dye adsorption method, the salt cleavage method, themethod of titration to neutralization and the iodine method.

However, from the description of the invention claimed in Europeanpatent No. 0,561,379, in particular from the measuring methods proposed,it emerges that the surface electrical charge in fact corresponds to theoverall ionic capacity of the semi-permeable membranes. Consequently,this European patent relates only to semi-permeable membranes with anoverall ionic capacity of greater than or equal to −30 μeq/g ofmembrane, but not to semi-permeable membranes with an overall ioniccapacity of very much less than −30 μeq/g of membrane, such as, forexample, the membrane manufactured by the company Hospal from acopolymer of acrylonitrile and of sodium methallyl sulphonate, which isknown under the trade name AN69.

Now, it is desirable not to modify the ionic capacity of membranes sincethis capacity gives rise to the adsorption and/or transfer of: certainproteins such as β2-microglobulin, inflammation mediators and complementfactors; lipids. This is particularly true for membranes with ahomogeneous, symmetrical structure.

Moreover, the Applicant has observed, on several examples, that amembrane which has an overall ionic capacity of greater than −30 μeq/gof membrane can lead to activation of the contact phase, whereas,conversely, a membrane with an overall ionic capacity which is markedlyless than −30 μeq/g of membrane cannot lead to activation of the contactphase.

Given the preceding observations, at the present time, a solution whichis satisfactory, in both medical and economic terms, for preventingactivation of the contact phase of blood or plasma which comes intocontact with a negatively-charged semi-permeable membrane does notappear to be known.

One aim of the invention is thus to solve the abovementioned problemwith apparatus for the treatment of blood or plasma by extra-corporalcirculation, comprising a semi-permeable membrane based onpolyacrylonitrile bearing fixed negative charges, which possesses twocharacteristics that have hitherto been considered antinomic, i.e.:

a negative overall ionic capacity, corresponding to an excess ofnegative charges, which simultaneously participates in thebiocompatibility of the membrane and is a factor for triggeringactivation of the contact phase, and

a capacity not to produce activation of the contact phase under normalconditions for a first use.

Another aim of the invention is to solve the above-mentioned problemwith sterilized apparatus for the treatment of blood or plasma byextra-corporal circulation, which has a capacity not to produceactivation of the contact phase under normal conditions for a first useand which is stable in storage. Lastly, another aim of the invention issterilized apparatus for the treatment of blood or plasma byextra-corporal circulation, which has a capacity not to produceactivation of the contact phase under normal conditions for a first useand which is ready to use, i.e. which does not require any specialmanipulation on the part of the user of the apparatus, such as a specialmanipulation for the purposes of preventing the adverse effects ofactivation of the contact phase.

To this effect, the invention proposes the use of a neutral or cationicpolymer for preventing activation of the contact phase of blood orplasma which comes into contact with a semi-permeable membrane based onpolyacrylonitrile bearing fixed negative charges, which is fitted inapparatus for the treatment of blood or plasma, by extra-corporalcirculation, according to which:

(1) the neutral or cationic polymer is combined with this semi-permeablemembrane, before or after formation of the membrane, and beforesterilization of the membrane;

(2) the overall ionic capacity of the semi-permeable membrane, based onpolyacrylonitrile bearing fixed negative charges and containing aneutral or cationic polymer, is negative;

(3) the overall ionic capacity of the semi-permeable membrane, based onpolyacrylonitrile bearing fixed negative charges and containing aneutral or cationic polymer, is, as an absolute value, not more than 10%less than the overall ionic capacity of the same semi-permeable membranewhich does not contain a neutral or cationic polymer; preferably, it is,as an absolute value, not more than 1% less than the overall ioniccapacity of the same semi-permeable membrane which does not contain aneutral or cationic polymer;

(4) the semi-permeable membrane containing a neutral or cationic polymercomplies, before sterilization, with one or other of the following twoelectrical characteristics:

the electrokinetic index “I” which is equal to Log₁₀ (|Z|/R), i.e. tothe logarithm, in base 10, of the ratio |Z|/R of the Zeta potential “Z”,as an absolute value, expressed in microvolts (or μV), of the surface ofthe membrane intended to come into contact with the blood or plasma, tothe electrical resistivity “R” of the membrane, expressed inohm.centimeters (or Ω.cm), is less than or equal to 0.8, or

the Zeta potential “Z” is positive and is between 0 and +15 mV (limitsnot included).

Preferably, the electrokinetic index “I” is less than or equal to 0.7and better still less than or equal to 0.6.

Preferably, when the Zeta potential is positive, it is strictly lessthan 15 mV and greater than or equal to I mV. Better still, the Zetapotential is greater than or equal to 6 mV and strictly less than 15 mV.

In order to obtain the electrokinetic index “I”:

the Zeta potential “Z” is calculated by measuring the potential “E”,expressed in volts, created by the flow of an electrolyte (10⁻²M NaCl),for example inside a bundle of hollow fibers. This potential “E”,measured between two Ag/AgCl electrodes connected to a high-impedancevoltmeter (Keithley 617), is linked to the Zeta potential “Z”, expressedin volts, by the Helmholtz-Smoluchowski law:$Z = \frac{4\pi \quad v\quad \lambda \quad E}{ɛ\quad P}$

P is the hydrostatic pressure giving rise to the flow of theelectrolyte, in millimeters of mercury (mmHg) (the ratio E/p is referredto as the flow potential),

v is the dynamic viscosity of the electrolyte, in Pascals,

λ is the actual electrical conductivity of the system in equilibriumwith the electrolyte (obtained by measuring the resistance with 10⁻²MNaCl) and is expressed in Siemens/m,

ε is the dielectric constant of the electrolyte, or the permittivity.

and the electrical resistivity “R”, expressed in Ω.cm, is deduced bymeasuring the electrical resistance of the semi-permeable membrane inequilibrium with an electrolyte (5×10⁻⁵M NaCl), using a Wheatstonebridge which functions with an alternating current (Wawetek generator,model 19, frequency 10 Hz).

The experimental determination of the electrical characteristics of thesemi-permeable membrane containing a neutral or cationic polymer must becarried out before sterilizing the membrane in the case of asterilization by irradiation. Otherwise, the obtained values arenonsensical and can not be directly in correlation with the amount ofneutral or cationic polymer to be used. In the other, less energeticmodes of sterilization, such as sterilization with ethylene oxide, thisexperimental determination can be carried out before or aftersterilization.

In the context of the invention, it is estimated that activation of thecontact phase is effective as soon as the maximum concentration ofkallicreins (KK) produced during the first 10 minutes of contact withblood or plasma exceeds 10 units of kallicreins (KK) per liter of bloodor plasma (10 UKK/l), taking into account the sensitivity of thechromogenic test used.

The term “semi-permeable membrane” is intended to refer to a flatmembrane or a bundle of hollow fibers. Consequently, the apparatus forthe treatment of blood or plasma by extra-corporal circulation generallycomprises two compartments separated by the semi-permeable membrane.

The expression “semi-permeable membrane based on polyacrylonitrilebearing fixed negative charges” is intended to refer to a semi-permeablemembrane consisting of polyacrylonitrile to which anionic functionalgroups are attached via covalent bonds, this negatively-chargedpolyacrylonitrile not being water-soluble.

The expression “neutral or cationic polymer combined (or incorporated)with the semi-permeable membrane” is intended to mean that this polymeris introduced:

either into the mass of the negatively-charged polyacrylonitrile (thisprocess is more particularly suitable for the neutral polymer)

or to the surface of the semi-permeable membrane, for example by placingthe membrane in contact with a solution containing the polymer (thisprocess is more particularly suitable for the cationic polymer) or byspraying with a solution containing the polymer.

In addition, the operating conditions for carrying out this combination(or incorporation) are designed to promote the presence of at least someof the neutral or cationic polymer at the surface of the semi-permeablemembrane (for example by promoted migration of the neutral polymer orionic bonding of the cationic polymer).

The term “swollen polymer” is intended to refer here to the membranehydrated to the content corresponding to the clinical use.

Surprisingly, it has been found that it is possible to preventactivation of the contact phase, which can occur fleetingly during theuse of apparatus for the treatment of blood or plasma by extra-corporalcirculation, by means of a semi-permeable membrane based onpolyacrylonitrile bearing fixed negative charges, by modifying theelectrical characteristics of the membrane, so that one or other of thefollowing two conditions are satisfied, before sterilization:

the electrokinetic index “I”, which is equal to Log₁₀ |Z|/R, is lessthan or equal to 0.8, or

the Zeta potential “Z” is positive and ranges between 0 and +15 mV(limits not included),

while at the same time substantially maintaining the overall ioniccapacity of the membrane, the modification of these electricalcharacteristics being obtained by combining this membrane with asuitable amount of neutral or cationic polymer, this combination beingcarried out:

firstly, before sterilization,

secondly, before or after formation of this membrane.

The prevention of activation of the contact phase has in particular beenobtained with semi-permeable membranes based on highlynegatively-charged polyacrylonitrile, reaching overall ionic capacitiesof less than or equal to −100 μeq/g of swollen polymer.

Surprisingly also, sterilization of the apparatus has no influence onits capacity to prevent activation of the contact phase.

Under the normal conditions of use of the apparatus according to theinvention, the known qualities of the semi-permeable membrane are keptintact when this same membrane contains a neutral or cationic polymer:for example, for a haemodialysis/haemofiltration membrane, thehaemocompatibility, the performance levels in terms of diffusive andconvective transfers, the capacity for adsorbing undesirable substances,etc, are kept intact.

In addition, interestingly, it has not been noticed adsorption of theanticoagulant agents, such as heparin, in a semi-permeable membrane,based on polyacrylonitrile bearing fixed negative charge, modifiedaccording to the present invention, in the case the sterilization iscarried out by irradiation.

According to the invention, the semi-permeable membrane based onnegatively-charged polyacrylonitrile, before combination with a neutralor cationic polymer, has a density of electronegative surface chargescorresponding to excesses of negative charges which can be detected, inparticular by the electrokinetic potential measurements (Zetapotential). The neutral or cationic polymer makes it possible to mask,at least partly, the electronegative charges present at the surface ofthe membrane. In the case of a cationic polymer, the negative charges onthe membrane are masked in particular by ionic bonding.

Preferably, the neutral polymer is water-soluble at room temperature(about 20° C.).

Preferably, the cationic polymer is water-soluble at room temperature(about 20° C.). However, a cationic polymer which is soluble in anorganic solvent, such as alcohols, may be suitable for the invention.

The neutral or cationic polymer must be able to withstand energeticsterilization, such as gamma irradiation. In other words, at least someof the polymer must remain intact and be able to mask, in the desiredmanner, some of the electronegative surface charges of the membrane.Moreover, the polymer fixed to the membrane and irradiated must notbecome toxic.

The weight-average molecular mass of the polymer is at least equal to10,000 daltons.

Advantageously, the weight-average molecular mass of the neutral polymeris greater than 40,000 daltons, preferably greater than 100,000 daltons,and the average molecular mass of the cationic polymer is greater than25,000 daltons, preferably greater than 100,000 daltons. These molecularmasses are measured by a light-scattering method. As polymers which aresuitable for carrying out the present invention, mention may be made, asneutral polymers, of polyvinylpyrrolidones (PVP) and polyethyleneglycols (PEG) of different molecular masses; as cationic polymers,mention may be made of hydrophilic cationic polymers capable of beingadsorbed onto a semi-permeable membrane which has an electronegativesurface charge density, such as polyamines, for examplepolyethyleneimines (PEI), diethylaminoethyl dextrans or DEAE dextrans,and polymers and copolymers containing one or several quaternaryammonium groups.

According to a preferred embodiment of the invention, the polymer iscationic. Preferably also, it is chosen from polyethyleneimines (PEI).

The amount of neutral or cationic polymer to be combined with themembrane depends on the electrical characteristics (Z, R, I) targetedand is variable depending on the chemical nature of the polymer, butdoes not exceed 10% of the mass of polyacrylonitrile. This amount isgenerally not more than 2% of the mass of polyacrylonitrile constitutingthe membrane in the case of the neutral polymer.

The amount of cationic polymer to be combined with the membrane ispreferably between about 1 and about 10 mg per m² of membrane intendedto be in contact with blood or plasma (this amount is very much lessthan 1% of the mass of polyacrylonitrile constituting the membrane).

The invention is particularly suitable for semi-permeable membranesbased on polyacrylonitrile bearing fixed negative charges which give ita high absolute value of overall ionic capacity, even afterincorporation of the neutral or cationic polymer.

Thus, the invention is particularly suitable for semi-permeablemembranes, based on polyacrylonitrile bearing fixed negative charges andcontaining a neutral or cationic polymer, which have an overall ioniccapacity of less than −30 μeq per g of swollen polymer (i.e. themembrane), preferably less than −50 μeq per g of swollen polymer, asmeasured by the standard ion-exchange method: by way of reference, theelectronegative polymer used to prepare the membrane made of AN69 has anoverall ionic capacity or density of negative charges equal to about−180 μeg per g of swollen polymer.

Advantageously, the semi-permeable membrane is a flat membrane or abundle of hollow fibers based on an acrylonitrile homopolymer orcopolymer and combined with a neutral or cationic polymer. As examplesof acrylonitrile copolymers, mention may be made of:

(1) a copolymer of acrylonitrile and of at least one anionic oranionizable monomer containing, where appropriate, units derived from atleast one other monomer containing olefinic unsaturation which iscapable of being copolymerized with acrylonitrile, or

(2) a copolymer of acrylonitrile and of at least one nonionic andnon-ionizable monomer containing, where appropriate, units derived fromat least one other monomer containing olefinic unsaturation which iscapable of being copolymerized with acrylonitrile.

Some of these polyacrylonitriles, as well as the various monomers whichcan be selected as starting materials, and their manufacture, aredescribed more fully in U.S. Pat. No. 4,545,910 reissued under U.S. Pat.No. Re. 34,239.

Among these polyacrylonitriles, those with which the invention isparticularly suitable are defined under (1) above. In particular, theinvention is particularly suitable for those for which the anionic oranionizable comonomer is olefinically unsaturated and bears anionicgroups chosen from sulphonate, carboxyl, phosphate, phosphonate andsulphate groups, and, even more particularly, when this comonomer issodium methallyl sulphonate.

The precise nature of the counterion for the anionic groups is notessential for the correct functioning of the invention.

A subject of the invention is also processes for manufacturing apparatusfor the treatment of blood or plasma by extra-corporal circulation, forpreventing activation of the contact phase and comprising asemi-permeable membrane in the form of a flat membrane or a bundle ofhollow fibers, based on polyacrylonitrile bearing fixed negative chargesand combined with a neutral or cationic polymer.

A first manufacturing process comprises the steps of:

preparing a solution consisting of:

at least one polyacrylonitrile bearing fixed negative charges,

at least one neutral polymer, in an amount adjusted in order to obtainthe four characteristics (1)-(4) above-mentioned,

at least one solvent for the said polyacrylonitrile and for the neutralpolymer,

optionally, at least one non-solvent for the polyacrylonitrile;

extruding this solution in order to form a hollow fiber or a flatmembrane;

simultaneously, in the case of the preparation of a hollow fiber, orafter extrusion in the case of the formation of a flat membrane,solidifying the membrane obtained by a phase inversion process bypartial or total contact of the extruded product with a liquid orgaseous fluid which is chemically inert with respect to the saidpolymers;

washing the flat membrane or the hollow fiber obtained;

optionally, treating the flat membrane or the hollow fiber withglycerol;

preparing a semi-permeable membrane from the flat membrane or assemblinga bundle of hollow fibers from the hollow fiber;

mounting the flat membrane or the bundle of hollow fibers in a case and,where appropriate, fixing caps to the case;

sterilizing the medical apparatus obtained.

When the polymer is cationic, it can be combined with the semi-permeablemembrane after the extrusion step for obtaining a hollow fiber or a flatmembrane, according to a second process comprising the steps of:

(a)preparing a flat membrane or a hollow fiber, which has optionallybeen treated with glycerol, by a conventional process from a solution ofpolyacrylonitrile bearing negative charges;

(b)assembling, in a conventional manner, the various components of theapparatus, in particular mounting the semi-permeable membrane or abundle of hollow fibers in a case and attaching the caps to this case;

(c)simultaneously or successively, optionally removing the glycerol fromthe semi-permeable membrane and preparing a solution containing thecationic polymer in dissolved form and bringing this solution intocontact with the surface of the semi-permeable membrane intended to beplaced in contact with blood, it being possible for step (c) to becarried out before or after step (b), the amount of cationic polymerbeing adjusted in order to obtain the four characteristics (1)-(4)above-mentioned,

(d)when the abovementioned step (c) has been carried out subsequent tostep (b), purging the apparatus of the solution containing the cationicpolymer;

(e)optionally, rinsing the semi-permeable membrane in order to removethe excess of non-bound cationic polymer and, optionally, retreating thesemi-permeable membrane with glycerol;

(f)sterilizing the medical apparatus.

Advantageously, the cationic polymer is water-soluble and the solutionin which this polymer is dissolved is aqueous.

In the case of a flat semi-permeable membrane, this membrane can becombined with a neutral or cationic polymer, preferably a cationicpolymer, using a spraying process comprising the following steps:

(a)preparing a flat membrane, which has optionally been treated withglycerol, from a solution of polyacrylonitrile bearing negative charges;

(b)simultaneously or consecutively, optionally removing the glycerolfrom the semi-permeable membrane and preparing a solution containing thecationic or neutral polymer in dissolved form and spraying this solutiononto the surface of the semi-permeable membrane intended to be placed incontact with blood, the amount of cationic or neutral polymer beingadjusted in order to obtain the four characteristics (1)-(4)above-mentioned,;

(c)assembling the various components of the apparatus, in particularmounting the semi-permeable membrane in a case and attaching the caps tothis case;

(d)optionally, rinsing the semi-permeable membrane in order to removethe excess of non-bound cationic polymer and, optionally, retreating thesemi-permeable membrane with glycerol;

(f)sterilizing the medical apparatus.

In addition, in the context of the above-mentioned processes formanufacturing apparatus for the treatment of blood or plasma byextra-corporal circulation, for preventing activation of the contactphase and comprising a semi-permeable membrane in the form of a flatmembrane or a bundle of hollow fibers, based on polyacrylonitrilebearing fixed negative charges and combined with a neutral or cationicpolymer, the amount of polymer, whether it is neutral or cationic, isadjusted so as to satisfy the following conditions:

the overall ionic capacity of the semi-permeable membrane, based onpolyacrylonitrile bearing fixed negative charges and containing apolymer (neutral or cationic), is negative;

the overall ionic capacity of the semi-permeable membrane, based onpolyacrylonitrile bearing fixed negative charges and containing apolymer (neutral or cationic), is, as an absolute value, not more than10% less than the overall ionic capacity of the same semi-permeablemembrane which does not contain polymer (neutral or cationic);preferably, it is, as an absolute value, not more than 1% less than theoverall ionic capacity of the same semi-permeable membrane whichcontains no polymer;

the electrical characteristics of the semi-permeable membrane containinga polymer (neutral or cationic) satisfies one or other of the followingtwo conditions:

the electrokinetic index “I” is equal to Log₁₀ (|Z|/R), i.e. to thelogarithm, in base 10, of the ratio |Z|/R of the Zeta potential “Z”, asan absolute value, expressed in microvolts (or μV), of the surface ofthe membrane intended to come into contact with the blood or plasma, tothe electrical resistivity “R” of the membrane, expressed inohm.centimeters (or Ω.cm), is less than or equal to 0.8, or

the Zeta potential “Z” is positive and is between 0 and +15 mV (limitsnot included).

Preferably, the electrokinetic index “I” is less than or equal to 0.7and better still less than or equal to 0.6.

Preferably, when the Zeta potential is positive, it is greater than orequal to 1 mV and strictly less than 15 mV.

Other operating conditions for preparing the semi-permeable membrane maybe found in U.S. Pat. No. 4,749,619 (gelation process) or in U.S. Pat.No. 4,056,467 (coagulation process).

Depending on the case, the sterilization technique which will be used,without any significant effect on the bonding between the neutral orcationic polymer and the semi-permeable membrane based onpolyacrylonitrile, may be sterilization by irradiation, in particular bygamma irradiation, or sterilization with ethylene oxide.

The abovementioned processes for manufacturing apparatus for thetreatment of blood or plasma by extra-corporal circulation have a majoradvantage: the apparatus obtained does not require any specificmanipulation on the part of the user, in particular during the phases ofrinsing and priming the apparatus, and the use of the apparatus by theuser is entirely identical to that of any apparatus of the same type.

Lastly, the invention relates to apparatus for the treatment of blood orplasma by extra-corporal circulation, which is sterilized and ready touse, for preventing activation of the contact phase and comprising asemi-permeable membrane, in the form of a flat membrane or a bundle ofhollow fibers, based on polyacrylonitrile bearing fixed negativecharges, characterized in that, before or after formation of thesemi-permeable membrane, and before sterilization, at least one neutralor cationic polymer is incorporated into the semi-permeable membrane, ina suitable amount such that:

the overall ionic capacity of the semi-permeable membrane, based onpolyacrylonitrile bearing fixed negative charges and containing aneutral or cationic polymer, is negative;

the overall ionic capacity of the semi-permeable membrane, based onpolyacrylonitrile bearing fixed negative charges and containing aneutral or cationic polymer, is, as an absolute value, not more than 10%less than the overall ionic capacity of the same semi-permeable membranewhich contains no neutral or cationic polymer;

the semi-permeable membrane based on polyacrylonitrile bearing fixednegative charges and containing a neutral or cationic polymer complieswith one or other of the following electrical characteristics:

the electrokinetic index “I” of the membrane containing a neutral orcationic polymer is less than or equal to 0.8, “I” being equal to thelogarithm, in base 10, of the ratio |Z|/R, where “Z” is the Zetapotential, as an absolute value, expressed in microvolts, of the surfaceof the membrane intended to come into contact with blood or plasma, andwhere “R” is the electrical resistivity of the membrane, expressed inohm.centimeters, or

the Zeta potential is positive and ranges between 0 and +15 mV (limitsnot included).

Preferably, the electrokinetic index “I” is less than or equal to 0.7and better still less than or equal to 0.6.

Preferably, when the Zeta potential is positive, it is greater than orequal to 1 mV and strictly less than 15 mV.

NON-LIMITING ILLUSTRATIVE EXAMPLES OF THE INVENTION Example 1

A minidialyser comprising 170 hollow fibbers made of AN69 (membraneconsisting of a copolymer of acrylonitrile and of sodium methallylsulphonate) was assembled. The blood compartment, or internalcompartment, is delimited by the interior of the fibbers and two caps,each fitted with an access tube, fixed to the ends of the case of theminidialyser.

Each fibber has the following dimensions:

inside diameter: 240 μm,

wall thickness: 50 μm,

length: 18 cm.

The inner surface area intended to come into contact with the blood orplasma is about 230 cm².

A solution of polyethyleneimine (PEI P, BASF, with an average molecularmass of 750,000 daltons) is prepared in a water/sodium chloride mixture(0.15 M NaCl).

The PEI concentration is 40 mg/l.

8 ml of this solution is circulated in the internal compartment of theminidialyser at a flow rate of 4 ml/min. 20 ml of aqueous 0.15 M NaClsolution for rinsing is then circulated in this same compartment at aflow rate of 4 ml/min.

Under these conditions, the amount of PEI bound to the membrane is about4 mg/m² (determined by measuring out the PEI leaving the minidialyser).

The fibbers thus treated are then retreated with glycerol by circulationof a glycerol/water mixture (60/40 by mass), in a proportion of 20 mlwith a flow rate of 4 ml/min, and purged with air in order to remove theexcess of mixture.

This minidialyser is sterilized with ethylene oxide.

Comment Regarding the Change in the Ionic Capacity:

The initial overall ionic capacity of the AN69 membrane is about −200μeq/g of swollen polymer.

If we consider the general formula of PEI —(CH₂—CH₂—NH)_(n)—, thiscontains about 23 μmol of amine groups per mg of PEI.

In the hypothesis in which all the amine groups are ionically linked tothe sulphonate groups of AN69 (which is not physically possible), asimple calculation shows that the reduction in the overall ioniccapacity of the membrane is negligible (about 0.8%).

Capacity of This Minidialyser to Prevent the Activation of the ContactPhase:

Prior to this test, the minidialyser is rinsed by circulating 20 ml of a0.15M NaCl solution at a flow rate of 2 ml/min.

A biological liquid, capable of stimulating the production ofkallicreins on contact with a negatively-charged surface membrane, wasprepared. The biological liquid used for the test consisted ofplatelet-poor human plasma, diluted to 5% in physiological salinesupplemented with citrate as anticoagulant (it is noted that theconditions of the test used are different from the conditions for usingapparatus for extra-corporal circulation of blood: the dilution rate isvery high, the liquid chosen is plasma and not blood, the plasma issupplemented with citrate, and thus acidified, whereas, in dialysis, theanticoagulant used is heparin. These test conditions are chosenintentionally since they stimulate and amplify the activation of thecontact phase). This liquid is circulated in an open circuit in theinternal compartment of the minidialyser at a flow rate of 2 ml/min for3 minutes. The plasmatic kallicreins were measured out in samples ofliquid taken after an interval of time by means of a standardchromogenic test, using the substrate S2302 from the company Biogenic.

As shown in Table 1, this treatment substantially modifies theelectrical characteristics of the membrane and has the effect ofinhibiting the activation of the contact phase.

TABLE 1 Electrical characteristics and level of activation of thecontact phase of the membranes AN69 and AN69 modified with PEI. ZetaElectro- Concentration potential Resistivity kinetic of kallicreinsMembrane mV ohm.cm index I formed, in U/l AN69 −70 312 1.98 70 AN69modi-  −1 312 0.5 <10 fied with PEI

Example 2

The solutions (2a and 2b) below are obtained using an extruder screw ata temperature of about 130° C. Table 2A gives the compositions of thesesolutions, expressed as a percentage by mass.

TABLE 2A Dralon L DMF PVP Solution a) b) 2-Butoxyethanol c) 2a 28%   51%21% 0 2b 28% 50.5% 21% 0.5% a) copolymer of acrylonitrile and of vinylacetate, sold under the name Dralon L by Bayer b) dimethylformamide c)polyvinylpyrrolidone K90 sold by the company Aldrich, with an averagemolecular mass equal to 360,000 daltons.

These solutions are extruded through a tubular die whose dimensions are1200/860/520 μm. The internal fluid is nitrogen. After cooling withambient air (20° C.), the heat-reversible gel obtained is washed withwater and diluted by a factor of 2 in the water at 60° C. The membraneobtained is treated with glycerol by immersing in a glycerol/watermixture (60/40 by mass).

The overall ionic capacity of the membrane obtained from the solution(2a) is about −20 μeq/g of swollen polymer and that of the membraneobtained from the solution (2b) is equal to about −19 μeq/g.

Minidialysers (identical to those described in Example 1) are made withthese membranes.

The minidialysers are sterilized by gamma irradiation (25/36 kGy).

The rinsing operation and the biological test (activation of the contactphase) are carried out according to the operating conditions given inExample 1.

Table 2B gives the results obtained as regards the electricalcharacteristics and the biological properties (activation of the contactphase).

TABLE 2B Electrical characteristics and level of activation of thecontact phase for membranes based on Dralon L which are modified or notby addition of PVP Zeta Electro- Concentration potential Resistivitykinetic of kallicreins Membrane mV ohm.cm index I formed, in U/l DralonL −39 5500 0.85 24 Dralon L −14 5830 0.38 <10 modified by addi- tion ofPVP

Example 3

A dialyser (trade name Crystal 4000, manufactured by Hospal), consistingof 57 parallel blood compartments separated by a flat AN69 membrane, hasa surface area capable of coming into contact with the blood of 1.53 m².This dialyser is made to undergo the following steps:

circulation in the blood compartment of 2 liters of physiological salineat a flow rate of 200 ml/min (ultrafiltration rate of 22 ml/min).

circulation in the blood compartment of 500 ml of a solution PEI with anaverage molecular mass of greater than 750,000 daltons, at aconcentration of 40 mg/l in distilled water and at a flow rate of 200ml/min (ultrafiltration rate of 22 ml/min).

rinsing by circulation in the blood compartment of 2 liters of aphysiological saline solution at a flow rate of 200 ml/min(ultrafiltration rate of 22 ml/min).

retreatment with glycerol by circulation in the blood compartment of Iliter of a glycerol/water solution (60/40 by mass) at a flow rate of 200ml/min (ultrafiltration rate of 22 ml/min).

The Zeta potential “Z” is calculated from the measurement of thepotential “E” according to the conditions given in the description.

Next, after purging with air, the dialyser is sterilized by gammairradiation (25 to 36 kGy).

After storage, and after rinsing for 10 minutes by circulation of anNaCl solution (0.15 M) at 200 ml/min, the dialyser is tested as regardsits capacity to generate kallicreins on contact with dilute plasma,according to the method described in Example 1 (the flow rate of thebiological liquid in the internal compartment of the dialyser is, inthis case, 100 ml/min).

TABLE 3 Zeta potential “Z” and level of activation of the contact phaseof the dialysers AN69 and AN69 modified with PEI Concentration of Zetapotential kallicreins formed Dialyser mV U/l Crystal 4000 −72 75 Crystal4000 +2.9 <10 treated with PEI

Example 4

A flat AN69 membrane (20 μm thickness) is treated by spraying with PEIwith an average molecular mass of greater than 750,000 daltons at aconcentration of 5 g/kg in a 60/40 by mass glycerol/water mixture. Theamount deposited is about 9 mg/m² of membrane.

With this membrane, a dialyser is assembled containing 39 parallel bloodcompartments separated by the flat AN69 membrane, such that the facetreated is the one which is in contact with the blood. The surface areaof the membrane intended to come into contact with the blood is about1.04 m².

The Zeta potential “Z” is calculated from the measurement of thepotential “E” according to the conditions given in the description. Theflow potential of such a dialyser is equal to +10 μV/mmHg, whereas thatof a dialyser of the same type without PEI is equal to −47 μV/mmHg.

Next, after sterilization by gamma irradiation (36 K Gy), the dialyseris tested as regards its capacity to generate kallicreins in contactwith dilute plasma, according to the method described in Example 1 (theflow rate of the biological fluid in the internal compartment of thedialyser is, in this case 100 ml/min).

TABLE 4 Zeta potential “Z” and level of activation of the contact phaseof the dialysers AN69 and AN69 modified with PEI Concentration of Zetapotential kallicreins formed Dialyser mV U/l AN69 −70 59 AN69 modifiedby 14.8 <10 spraying with PEI

Example 5

A dialyser comprising about 8500 hollow AN69 fibers was assembled. Thesurface area of the membrane intended to come into contact with blood orplasma is about 1.34 m².

200 ml of a solution containing 5 g/kg of PEI (average molecular mass ofgreater than 750,000 daltons) are prepared in a 60/40 by massglycerol/water mixture.

This solution is made to circulate in the internal compartment of thedialyser in an open circuit at a flow rate of 200 ml/min. The bloodcompartment, or internal compartment, is delimited by the interior ofthe fibers and two caps each fitted with an access tube, which areattached to the ends of the dialyser case. The Zeta potential “Z” iscalculated from the measurement of the potential “E” according to theconditions given in the description. The flow potential measured withthis dialyser is equal to +2.7 μV/mmHg (−22 μV/mmHg in the case of adialyser of the same type without PEI).

Next, after sterilization by gamma irradiation (36 K Gy), the dialyseris tested as regards its capacity to generate kallicreins on contactwith dilute plasma, according to the method described in Example 1.

After rinsing for 10 minutes by circulation of an NaCl solution (0.15 M)at 200 ml/min, the dialyser is subjected to the biological testdescribed in Example 1 (the flow rate of the biological fluid in theinternal compartment of the dialyser is, in this case, 100 ml/min).

Table 5 below gives the results of the various measurements carried outand the level of activation of the contact phase achieved.

Concentration of Zeta potential kallicreins formed Dialyser mV U/l AN69−72 70 AN69 modified 9.3 <10 with PEI

The overall ionic capacity of the hollow fibbers in AN69, without PEI,is 174 μeq/g of swollen polymer and the one of the hollow fibers inAN69, modified by PEI, is 163 μeq/g of swollen polymer.

This invention can be used with any type of apparatus which incorporatesa semi-permeable membrane for the treatment of blood or plasma.

The attached FIGURE is a vertical cross sectional view of a preferredembodiment of apparatus of the invention but is merely intended to beexemplary and limiting of the invention.

DETAILED DESCRIPTION OF THE FIGURE

The FIGURE represents a membrane exchanger comprising two compartmentswhich are separated by a semi-permeable membrane composed of a(partially represented) bundle 1 of semi-permeable hollow fibers. Thebundle 1 is secured within a tubular housing 2 at both ends by means oftwo disks 3,4 of potting material. The disks 3,4 tie up the fiberstogether and they delimit between them within the housing 2 afluid-tight compartment to which two pipes 5,6 give access, which areperpendicular to the longitudinal axes 9 of the housing 2. Two end caps7,8 are respectively secured at the ends of the housing 2. Each end cap7,8 comprises an axial access pipe 9,10. The blood compartment of thisexchanger is composed of the lumens of the hollow fibers and of theinner space delimited between the end caps 7,8 and the disks 3,4 ofpotting material.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The preceding preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

The entire disclosure of all applications, patents and publications,cited above, and of French application 9716732, filed Dec. 24, 1997, arehereby incorporated by reference.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A method for preventing activation of the contactphase of blood or plasma which comes into contact with a semi-permeablemembrane based on polyacrylonitrile bearing fixed negative charges,which is fitted in apparatus for the treatment of blood or plasma, byextra-corporal circulation, said method comprising contacting blood orplasma with said semi-permeable membrane, wherein: a neutral or cationicpolymer is combined with said semi-permeable membrane before or afterformation of the membrane, and before sterilization of the membrane; theoverall ionic capacity of the semi-permeable membrane, based onpolyacrylonitrile bearing fixed negative charges and containing aneutral or cationic polymer, is negative; the overall ionic capacity ofthe semi-permeable membrane, based on polyacrylonitrile bearing fixednegative charges and containing a neutral or cationic polymer, is, as anabsolute value, not more than 10% less than the overall ionic capacityof the same semi-permeable membrane without a neutral or cationicpolymer; and the semi-permeable membrane based on polyacrylonitrilebearing fixed negative charges and containing a neutral or cationicpolymer complies, before sterilization, with the following electricalcharacteristic: the Zeta potential “Z” is positive and ranges in between0 and +15 mV.
 2. A method according to claim 1, characterized in thatthe Zeta potential “Z” is positive and is greater than or equal to +1 mVand less than +15 mV.
 3. A method according to claim 1, characterized inthat the Zeta potential “Z” is positive and is greater than or equal to+6 mV and less than +15 mV.
 4. A method according to one of claims 1 to5, characterized in that the overall ionic capacity of thesemi-permeable membrane based on polyacrylonitrile bearing fixednegative charges and containing a neutral or cationic polymer is lessthan −30 μeq/g of swollen polymer.
 5. A method according to claim 4,characterized in that the overall ionic capacity of the semi-permeablemembrane based on polyacrylonitrile bearing fixed negative charges andcontaining a neutral or cationic polymer is less than −50 μeq/g ofswollen polymer.
 6. A method according to claim 1, characterized in thatthe overall ionic capacity of the semi-permeable membrane based onpolyacrylonitrile bearing fixed negative charges and containing aneutral or cationic polymer is, as an absolute value, not more than 1%less than the overall ionic capacity of the same semi-permeable membranewhich does not contain a neutral or cationic polymer.
 7. A methodaccording to claim 1, characterized in that the polymer is neutral andhas an average molecular mass of greater than 40,000 daltons.
 8. Amethod according to claim 7, characterized in that the neutral polymerhas an average molecular mass of greater than 100,000 daltons.
 9. Amethod according to claim 7, characterized in that the neutral polymeris chosen from the group consisting of polyvinylpyrrolidones andpolyethylene glycols.
 10. A method according to one of claim 1,characterized in that the polymer is cationic and has an averagemolecular mass of greater than 25,000 daltons.
 11. A method according toclaim 10, characterized in that the cationic polymer has an averagemolecular mass of greater than 100,000 daltons.
 12. A method accordingto claim 10, characterized in that the cationic polymer is chosen frompolyamines.
 13. A method according to claim 12, wherein the cationicpolymer is a polyethyleneimine.
 14. A method according to claim 10,characterized in that the cationic polymer is chosen fromdimethylaminoethyl dextrans.
 15. A method according to claim 1,characterized in that the polyacrylonitrile constituting thesemi-permeable membrane is a copolymer of acrylonitrile and of at leastone anionic or anionizable monomer containing olefinic unsaturationwhich is capable of being copolymerized with acrylonitrile.
 16. A methodaccording to claim 15, characterized in that the polyacrylonitrile is acopolymer of acrylonitrile and of an olefinically unsaturated anionic oranionizable comonomer bearing sulphonate, carboxyl, phosphate,phosphonate and sulphate groups.
 17. A method according to claim 15,characterized in that the comonomer is sodium methallyl-sulphonate. 18.A method according to claim 1, characterized in that thepolyacrylonitrile constituting the semi-permeable membrane is acopolymer of acrylonitrile and of at least one nonionic andnon-ionizable monomer containing olefinic unsaturation which is capableof being copolymerized with acrylonitrile.
 19. A process formanufacturing apparatus for the treatment of blood or plasma byextra-corporal circulation, for preventing activation of the contactphase and comprising a semi-permeable membrane, in the form of a flatmembrane, based on polyacrylonitrile bearing fixed negative charges,characterized in that it comprises the following steps: (a)preparing aflat membrane, which has optionally been treated with glycerol, from asolution of polyacrylonitrile bearing negative charges; (b) optionallyremoving the glycerol from the semi-permeable membrane and preparing asolution containing the cationic or neutral polymer in dissolved formand spraying said solution onto the surface of the semi-permeablemembrane intended to be placed in contact with blood; (c)mounting thesemi-permeable membrane in a case and attaching the caps to this case;(d)optionally, rinsing the semi-permeable membrane in order to removethe excess of non-bound cationic polymer and, optionally, treating orretreating the semi-permeable membrane with glycerol; (f)sterilizing themedical apparatus; and in that the amount of cationic or neutral polymeris adjusted such that: the overall ionic capacity of the semi-permeablemembrane, based on polyacrylonitrile bearing fixed negative charges andcontaining a cationic or neutral polymer, is negative; the overall ioniccapacity of the semi-permeable membrane, based on polyacrylonitrilebearing fixed negative charges and containing a cationic or neutralpolymer, is, as an absolute value, not more than 10% less than theoverall ionic capacity of the same semi-permeable membrane whichcontains no cationic or neutral polymer; the semi-permeable membranebased on polyacrylonitrile bearing fixed negative charges and containinga cationic or neutral polymer complies, before sterilization, with thefollowing electrical characteristic: the Zeta potential “Z” is positiveand ranges in between 0 and +15 mV.
 20. An article of manufacture forthe treatment of blood or plasma by extra-corporal circulation, which issterilized and ready to use, for preventing activation of the contactphase and comprising a semi-permeable membrane, in the form of a flatmembrane or a bundle of hollow fibers, based on polyacrylonitrilebearing fixed negative charges, characterized in that, before or afterformation of the semi-permeable membrane, and before sterilization, atleast one neutral or cationic polymer is incorporated into thesemi-permeable membrane, in a sufficient amount such that: the overallionic capacity of the semi-permeable membrane, based onpolyacrylonitrile bearing fixed negative charges and containing aneutral or cationic polymer, is negative; the overall ionic capacity ofthe semi-permeable membrane, based on polyacrylonitrile bearing fixednegative charges and containing a neutral or cationic polymer, is, as anabsolute value, not more than 10% less than the overall ionic capacityof the same semi-permeable membrane which contains no neutral orcationic polymer; the semi-permeable membrane based on polyacrylonitrilebearing fixed negative charges and containing a neutral or cationicpolymer complies, before sterilization, with the following electricalcharacteristic: the zeta potential is positive and ranges in between 0and +15 mV.
 21. A process for manufacturing apparatus for the treatmentof blood or plasma by extra-corporeal circulation, for preventingactivation of the contact phase and comprising a semi-permeable membranein the form of a flat membrane or a bundle of hollow fibers, based onpolyacrylonitrile bearing fixed charges, characterized in that itcomprises the following steps: preparing a solution comprising: at leastone polyacrylonitrile bearing fixed negative charges, at least oneneutral polymer, at least one solvent for said polyacrylonitrile and forthe neutral polymer, optionally, at least one non-solvent for thepolyacrylonitrile; extruding the solution in order to form a hollowfiber or a flat membrane; simultaneously with extrusion, in the case ofa the preparation of a hollow fiber, or after extrusion in the case ofthe formation of a flat membrane, solidifying the membrane obtained by aphase inversion process by partial or total contact of the extrudedproduct with a liquid or gaseous fluid which is chemically inert withrespect to the polymers; washing the flat membrane or the hollow fiberobtained; optionally, treating the flat membrane or the hollow fiberwith glycerol; preparing a semi-permeable membrane from the flatmembrane or assembling a bundle of hollow fibers from the hollow fiber;mounting the flat membrane or the bundle of hollow fibers in a case andoptionally fixing caps to the case; sterilizing the medical apparatusobtained; and in that the amount of neutral polymer is adjusted suchthat: the overall ionic capacity of the semi-permeable membrane, basedon polyacrylonitrile bearing fixed negative charges and containing aneutral polymer, is negative; the overall ionic capacity of thesemi-permeable membrane, based on polyacrylonitrile bearing fixednegative charges and containing a neutral polymer, is an absolute value,not more than 10% less than the overall ionic capacity of the samesemi-permeable membrane without a neutral polymer; and thesemi-permeable membrane based on polyacrylonitrile bearing fixednegative charges and containing a neutral polymer complies, beforesterilization, with the following electrical characteristic: the Zetapotential “Z” is positive and ranges in between 0 and +15 mV.
 22. Aprocess for manufacturing apparatus for the treatment of blood or plasmaby extra-corporeal circulation, for preventing activation of the contactphase and comprising a semi-permeable membrane, in the form of a flatmembrane or a bundle of hollow fibers, based on polyacrylonitrilebearing fixed negative charges, characterized in that it comprises thefollowing steps: (a) preparing a flat membrane or a hollow fiber, whichhas optionally been treated with glycerol, from a solution ofpolyacrylonitrile bearing negative charges; (b) mounting thesemi-permeable membrane or a bundle of hollow fibers in a case andattaching caps to said case; (c) optionally removing the glycerol fromthe semi-permeable membrane and preparing a solution containing thecationic polymer in dissolved form and bringing said solution intocontact with the surface of the semi-permeable membrane intended to beplaced in contact with blood, it being possible for step(c) to becarried out before or after step (b); (d) when step (c) has been carriedout subsequent to step (b), purging the apparatus of the solutioncontaining the cationic polymer; (e) optionally, rinsing thesemi-permeable membrane in order to remove excess of non-bound cationicpolymer and, optionally, treating or retreating the semi-permeablemembrane with glycerol; (f) sterilizing the medical apparatus; and inthat the amount of cationic polymer is adjusted such that; the overallionic capacity of the semi-permeable membrane, based onpolyacrylonitrile bearing fixed charges and containing a cationicpolymer, is negative; the overall ionic capacity of the semi-permeablemembrane, based on polyacrylonitrile bearing fixed negative charges andcontaining a cationic polymer, is, as an absolute value, not more than10% less than the overall ionic capacity of the same semi-permeablemembrane which contains no cationic polymer; the semi-permeable membranebased on polyacrylonitrile bearing fixed negative charges and containinga cationic polymer complies, before sterilization, with the followingelectrical characteristic: the Zeta potential “Z” is positive and rangesin between 0 and +15 mV.